THE NATURAL AND UNNATURAL HISTORIES OF XENOPUS LAEVIS
Xenopus share a distinctive morphology and habitat. Though believed to have originated from terrestrial anurans (aquatic tadpoles, terrestrial adults), Xenopus is entirely aquatic. Migration across land from pond to pond has been observed in present day species but is limited in distance and time of year (rainy season) because, out of water, the frogs will dessicate within a day and die. In appearance these frogs are flattened dorsoventrally with dorsally (upwards) facing eyes as adults. Three toes of the hind limbs are clawed and a line of specialized sensory organs (the lateral line organs or "stitches") encircles the back and the eyes.
Figure 2, Distinctive features of Xenopus laevis. The back and eyes are encircled by distinctive "stitches" that contain the lateral line organs. Several digits of the hind foot carry black claws.
The most extensively examined Xenopus is laevis; all of the laboratory studies on this species use the sub-species laevis laevis from South Africa. What we know about the natural history of laevis is largely the result of talented and well-trained South African zoologists. An interest in endemic (native) species was natural for early zoologists; the isolation that resulted from government policies (apartheid) deepened interest in native plants and animals; South Africa, for example, is home to a unique floral kingdom. However, the prevalence of laevis (whose common name is platanna or platie on account of being flattened) and its uncharismatic appearance somewhat diminished its potential as an exotic. We were ourselves very surprised at the complex vocal repertoire used by these simple-appearing animals. Nonetheless, a number of useful descriptions of habitat and habits were published beginning at the turn of the 20th century. These proved particularly useful when the species was adopted for the laboratory first by endocrinologists and then by developmental biologists.
Before there were only mice, zoologists found it natural to work on a very large variety of species, each of which might have specific advantages for addressing a particular question (for a discussion of this approach in neuroethology see Eve Marder's 2002 essay in Nature). Developmental biologists such as Thomas Hunt Morgan studied amphibians because the eggs and early embryos were large; experimental manipulations were thus relatively easy and development outside of the uterus or egg meant that results could be tracked throughout development (one didn't have to wait for birth or hatching). The drawback was that most amphibians have a strong circannual cycle; getting embryos outside of the normal breeding season was difficult or impossible and experiments were thus limited to only certain parts of the year. (This difficulty was, in part, responsible for the adoption of the chick as a developmental model).
When Lancelot Hogben arrived at the University of Cape Town to take up the chair of Zoology in 1927 he inadvertently solved this problem (Hogben, 1998; see Gratzer, 1998). Hogben, an irrascible but talented scientific polymath was, at this point in his career, an endocrinologist (he had, for example, isolated melanocyte stimulating hormone). Having, as was his wont, burned his bridges at his previous job (McGill), he set out - with travel funds advanced by Julian Huxley- for Cape Town where "the fauna and climate were uniquely favorable for biological investigation" (Hogben, p. 90). Among Hogben's interests at the time was the endocrine role of the pituitary and his son Adrian states (xii) that his father believed that Xenopus would become for endocrinology what Drosophila was for genetics. A byproduct of this interest was the Hogben pregnancy test, the first well documented assay for human pregnancy. This test - as modified in South Africa by Zwarenstein - comprised the injection of an extract of urine from pregnant women into female X. laevis; subsequent oviposition indicates the presence of human chorionic gonadotrophin (HCG; still the most useful indicator of early pregnancy). HCG also stimulates sexual behavior and sperm release in male Xenopus(see Kelley and Pfaff, 1979). Once HCG became commercially available, it was possible to produce embryos at any time of year and X. laevis became a very popular experimental system for developmental biologists. John Gurdon, for example (Gurdon et al., 1975), was the first to produce a cloned vertebrate using X. laevis and he used the system to show that a somatic nucleus contains all of the relevant genetic instructions for creating a new organisms (i.e. DNA is not discarded or permanently inactivated even in highly specialized cells). Gurdon was also instrumental in developing the Xenopus oocyte as an expression (translation) system for foreign genes. More recently, X. laevis has been instrumental in uncovering molecules used in early embryonic induction and is the only vertebrate system in which novel genes for this process have been identified.
Some further work on endocrinology of Xenopus was carried on by Emil Witschi and his colleague K. Mikamo in the 1950's and 60's. They established, for example, that it is possible to sex reverse X. laevis embryos by rearing them in a solution of estradiol; the result are all female tadpoles half of which, when mated to males, produce only male offspring. This set of experiments set up the prevalent hypothesis for sex determination in Xenopus (in which the sex chromosomes cannot be distinguished from autosomes by morphology): the genotype of males is zz and that of females is zw. We have recently used this method to sex reverse X. tropicalis embryos because an all female population will be useful for genetic studies (the nuclei of activated eggs can be made to fuse using pressure thus producing homozygotes). How estradiol converts the developing gonadal primordium into an ovary is not known (see Kelley, 1996, for discussion). Using Xenopus borealis Tony Blackler established that the decision of a primordial germ cell to become an egg or a sperm cell is determined by the genetic sex of the gonad into which it migrates and not by its intrinsic genetic makeup (zz, male or zw, female). A review of sexual differentiation in Xenopus laevis is the topic of Kelley, 1996.
Xenopus laevis remained a subject of Ph.D. theses on reproductive behavior in South Africa. John Hutchison studied the neural basis of the clasping reflex before leaving for Cambridge to work on birds. Mike Picker described male calls and their role in attracting females before turning to his current studies on beetles. Perhaps the most bizarre adoption of X. laevis was by W. Russell at Oxford. His motivation for the study of sexual behavior was an experimental test of ideas advanced by Sigmund Freud. The result was one of the earlier descriptions of reproductive behavior including several novel male calls and the female release call, ticking. Since these studies predated the use of sonagrams for sound analysis, it is difficult to definitively match his onamonopoetic descriptions, male "sawing" for example, to the vocal behaviors that we are currently investigating. Another difficulty (one we have only recently solved; Tobias et al., 2002) is that there are no body movements associated with sound production and one cannot tell which frog is vocalizing. The very strong focus on male vocalizations resulting from Darwin's sexual selection hypothesis (Darwin, 1891) led most investigators to underestimate female songs.
Nowadays Xenopus laevis of either sex and at any age can be easily obtained from commercial suppliers. Tadpole development was very usefully divided into 66 stages based on external morphology (The "Normal Table" developed by Nieuwkoop and Faber, 1956 but recently reprinted). Depending on rearing conditions,tadpoles complete metamorphosis in 2 - 3 months. We have divided the post-metamorphic development of males into stages of masculinization based on laryngeal characteristics and standardized to age and weight (Tobias et al., 1991a). Males are typically sexually mature at 1year and females at 2yrs though optimal rearing conditions will bring animals along faster. Nonetheless the long period to sexual maturity for laevis females is one reason that tropicalis has been embraced with such fervor by developmental biologists interested in doing genetic studies. The species (laevis) is long lived (~30yrs) in the laboratory but older females have atretic eggs and cannot be used for breeding. A description of the rearing conditions in our Xenopus colonies and of some disorders we have encountered can be found on the Kelley laboratory web site.
In terms of natural history, X. laevis inhabit silt-filled ponds preferentially and thus usually cannot be observed (though see Elepfandt, 1996, for a description of a small population in a clear pool near Johannesburg). The breeding season depends on temperature and rainfall (Kalk, 1960). In Cape Town breeding occurs during the South African winter months. In field studies carried out in 1995 and 1997,Martha Tobias recorded calling from mid-June until late November. The tadpoles are herbivorous and the adults carnivorous. In 1997 we studied populations in two attached ponds (water sources for a golf course) near Cape Town. The upper pond was inhabited only by tadpoles and juveniles while the adults congregated in the lower pond. When other sources of food in a pond are depleted the adults will eat the young. Mike Picker had argued (1996) that this mechanism has favored the mating of different Xenopus species trapped together in a small pond but how often this actually occurs in the wild is not known.